Capillary array electrophoresis apparatus and method of separating and analyzing specimen

ABSTRACT

At a wall constituting a space for a thermostatic oven in an electrophoresis apparatus, a capillary array attachment portion is formed which permits attachment of a plurality of capillary arrays having different length. Thereby, a selected capillary array constituted by collecting a plurality of capillaries can be easily attached to the electrophoresis apparatus depending on measurement purpose.

The present application is a continuation of application Ser. No.09/852,029, filed May 10, 2001, now U.S. Pat. No. 6,936,152, the entiredisclosure of which is incorporated herein by reference, and whichclaims foreign priority from Japanese application 2000-147495, filed May15, 2000.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a capillary array electrophoresisapparatus and a method of separating and analyzing specimen which can beused for separating and analyzing specimen such as DNA and protein.

2. Conventional Art

An application technology in which an array is constituted by combininga plurality of capillaries, an electrophoresis medium and a sample to beseparated or analyzed are supplied to the respective capillaries andmoved therethrough to thereby separate and analyze the object sample iswell known, wherein a sample such as DNA and protein marked by afluorescent material is supplied to the capillaries. Such applicationtechnology is, for example, disclosed in U.S. Pat. Nos. 5,366,608,5,529,679, 5,516,409, 5,730,850, 5,790,727, 5,582,705, 5,439,578 and5,274,240. In view of a through-put of the separation and analysis, itis much more advantageous to use electrophoresis with multi capillariesrather than electrophoresis with a flat plate gel.

A capillary array electrophoresis apparatus is basically constituted bysuch as a capillary array, an excitation light system including a laserbeam source, a light receiving optical system which detects fluorescenceand a voltage application unit which causes electrophoresis. In suchcapillary array electrophoresis apparatus, the capillary array isconstituted by aligning a plurality of capillaries in a plane shape, anda laser beam is irradiated to the capillaries which are filled with asample fluorescent sample) marked by a fluorescent material in paralleldirection with the capillary aligning direction, then, through the lensaction of the capillaries the laser beam is condensed and the laser beamis irradiated to the fluorescent sample in all of the capillaries whenthe laser beam is irradiated, the fluorescent sample emits fluorescent.Through detection by the light receiving optical system of thefluorescent emitted from the fluorescent sample in a directionsubstantially perpendicular to the laser beam irradiation direction, themeasurement of the sample is performed.

Since time required for electrophoresis, separation and resolutiondiffer depending on molecular weight and molecular structure of theobject sample, it is necessary to change the length of electrophoresispassage depending on the object sample. Therefore, it becomes necessaryto selectively dispose several kinds of capillary arrays in a space of athermostatic oven.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a capillary arrayelectrophoresis apparatus which permits easy handling, when exchanging aconsumable capillary array therein.

The present invention provides a capillary array electrophoresisapparatus comprising, a thermostatic oven which permits temperatureadjustment and includes a space which can accommodate a plurality ofcapillary arrays of different length exchangeably, a capillary arraywhich is selected depending on an object sample and is disposed in thespace; means for supplying the object sample into capillaries in thecapillary array from one end of the capillary array; means for supplyingan electrophoresis medium into the capillaries from the other end of thecapillary array; means for irradiating light beam to the object sampleexisting within the capillaries in the capillary array at the out sideof the thermostatic oven and for causing emission of fluorescencetherefrom; and means for detecting the fluorescence. Thereby, acapillary array can be selected depending on an object sample to beseparated and analyzed and a capillary array can be easily attachedwithin a space of a thermostatic oven.

Further, the present invention provides a capillary arrayelectrophoresis apparatus in which a plurality of fans each havingdifferent air suction and air discharge directions are disposedsubstantially most separate positions in the space of the thermostaticoven to agitate the air therein. With such arrangement of the pluralityof fans the air within the space in the thermostatic oven is desirablyagitated without vibrating the capillary array.

Still further, the present invention is to provide a capillary arrayelectrophoresis apparatus comprising a first syringe having apredetermined volume, a second syringe having a smaller volume than thatof the first syringe and a pump device which injects under pressure anelectrophoresis medium to the first syringe and further injects underpressure the electrophoresis medium of a predetermined amount from thefirst syringe to the second syringe through a check value, wherein thevolume of the second syringe is determined in view of the amount of theelectrophoresis medium consumed substantially in an one time separationand analysis. Thereby, with the provision of such electrophoresis mediumsupply system a series of operation from recharging of the medium,supplying of the sample and to separation and analysis of the sample canbe automated.

Still further, the present invention is to provide a capillary arrayelectrophoresis apparatus in which major elements in the fluorescentdetection means are substantially arranged on one plane face and therespective capillaries at the irradiation and detection portion in thecapillary array are aligned so as to cross to the one plane face.Through such arrangement of the capillary array and the optical system,a compact separation and analysis system can be obtained.

Still further, the present invention is to provide a capillary arrayelectrophoresis apparatus in which the sample is supplied to the one endof the capillary array from the bottom portion in the space of thethermostatic oven, the other end of the capillary array containing thesample subjected to electrophoresis is projected from the side portionof the space and the laser beam is irradiated onto the projectedcapillary array, thereby, fluorescence is outputted. With thisarrangement of the capillary array a compact electrophoresis apparatuscan be obtained.

Still further, the present invention is to provide a capillary arrayelectrophoresis apparatus in which an array plane face constituting thedetection portion of the capillary array is arranged to be substantiallyin parallel with the laser beam. With such arrangement of the capillaryarray and the optical system a compact electrophoresis apparatus can beobtained.

Still further, the present invention is to provide a method ofseparating and analyzing sample in which a capillary array is disposedin a space of a thermostatic oven which permits temperature adjustmentand includes the space which can accommodate a plurality of capillaryarrays of different length exchangeably, an object sample is suppliedinto capillaries in the capillary array from one end of the capillaryarray; an electrophoresis medium is supplied into the capillaries fromthe other end of the capillary array so as to fill the capillaries;laser beam is irradiated to the object sample existing within thecapillaries in the capillary array at a range of the capillary arrayprojecting from the space of the thermostatic oven; and fluorescenceemitted by the laser beam irradiation is detected.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an outlook perspective view of a capillary arrayelectrophoresis apparatus according to the present invention;

FIG. 2 is a perspective view of a thermostatic oven in FIG. 1;

FIG. 3 is a perspective view of the thermostatic oven in FIG. 2, whenthe door thereof is opened;

FIG. 4 is a perspective view showing a relationship between a bottomstructure and a capillary array holder in the thermostatic oven as shownin FIG. 3;

FIG. 5 is a perspective view of the capillary array holder as shown inFIG. 4, when seen from the back face thereof;

FIGS. 6A and 6B are schematic cross sectional views showing innerstructure of the thermostatic oven as shown in FIG. 2;

FIGS. 7A through 7D are schematic diagrams showing air flow directionsof two fans disposed in the thermostatic oven;

FIG. 8 is a schematic diagram showing a structure of major portions ofan electrophoresis apparatus according to the present invention;

FIG. 9 is a perspective view showing a structure of a separator whichaligns and holds respective capillaries in a capillary array;

FIGS. 10A and 10B are an upper face view and a side face view showing astructure of a separator holder for holding the separator as shown inFIG. 9 on a wall of the thermostatic oven;

FIG. 11 is a schematic diagram showing an imaginary attachment state ofa plurality of capillary arrays having different length in thethermostatic oven;

FIG. 12 is a schematic diagram for explaining laser beam irradiation andfluorescence detection in a capillary array electrophoresis apparatusaccording to the present invention;

FIG. 13 is a exploded view showing a structure of an irradiation anddetection portion in a capillary array used in the present invention;

FIG. 14 is a schematic diagram for explaining a gel pump systemaccording to the present invention;

FIG. 15 is a detailed cross sectional view of a connection portionbetween the gel pump system and the capillary array as shown in FIG. 14;

FIG. 16 is a connection diagram showing an example of methods ofmonitoring capillary current;

FIG. 17 is a block diagram for controlling a thermostatic oven;

FIG. 18 is a flow chart for explaining sequence of gel injection intocapillary array by the gel pump system; and

FIGS. 19A and 19B are schematic diagrams for explaining a mechanismwhich automatically stops generation of laser beam, when the operationof the capillary array electrophoresis apparatus according to thepresent invention is stopped.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinbelow, embodiments of the present invention will be explained indetail with reference to the drawings.

FIG. 1 shows a state in which a thermostatic oven 102 is detached from aDNA sequencer frame 101. A DNA sequencer includes, other than thethermostatic oven 102, a gel pump unit 103 which recharges and exchangesgel polymer working as a separation medium into capillaries and airradiation and detection portion 104 which irradiates such as laserbeam onto the capillary array and detects fluorescence therefrom, andfurther includes an auto sampler 105 for a continuous measurement.

When attaching and detaching the thermostatic oven 102 to the frame 101for assembling and maintenance thereof, it is desirable that a correctpositional relationship is always kept with the frame 101. The capillaryarray is to be attached to the thermostatic oven 102, thus if thepositional relationship of the thermostatic oven 102 with respect to theframe 101 is not kept, the positional relationship with the auto sampler105 is lost which requires a mechanical correction in a highpossibility. In the present invention, guide pins 106 and 107 areprovided for the frame 101 so as to maintain a correct positionalrelationship with the thermostatic oven 102.

FIG. 2 is a view of the thermostatic oven 102 seen from the back facethereof. On a reference plate of the back face guide holes 202 and 203are formed at the positions corresponding to the guide pins 107 and 106as shown in FIG. 1. When a positional accuracy for fitting between thesepins and holes is determined smaller than the tolerance which isrequired when attaching and detaching the thermostatic oven 102, acorrect positional relationship can be maintained. The requiredpositional accuracy is determined either by a positional accuracyrequiring no correction or by a positional accuracy which permitscorrection by a software. Herein, when a positional relationship is keptat the time of attachment, the thermostatic oven 102 is secured to theframe 101 by stationary screws 201. It is preferable to use a pluralityof stationary screws 201.

Further, the thermostatic oven 102 in the present embodiment uses aPeltier element as a heat source for heating and cooling which permits,other than a set temperature more than 50° C. which is used in a normalDNA sequencer, to set a temperature below a room temperature. At theback face of the thermostatic oven a heating and cooling device isprovided, wherein a heat radiation fin for a Peltier unit 204 isprovided and with a Peltier heat radiation fan 205 a heat exchangeefficiency is enhanced.

FIG. 3 shows a view when the door of the thermostatic oven 102 isopened. A packing 301 is disposed around the door, when the door islocked by a lock 302 while pressing the packing 301, a close contactbetween the thermostatic oven 102 and the door is ensured, thereby, anair flow therebetween is prevented. Thus, a temperature distribution andvariation within the thermostatic oven 102 are suppressed small.

At an attachment portion 307 of the capillary array it is necessary tomaintain a relative positional relationship between the attachedcapillary array and the auto sampler, therefore, an elastic packing cannot be used there, thus an air flow therethrough can not be kept zero.When the apparatus is used under an environment of high temperature andhigh humidity and the control temperature in the thermostatic oven 102is lower than the room temperature, it is possible that such as steamcontained in the originally existing air inside the thermostatic oven102 and steam newly entered into the thermostatic oven 102 through airflow therebetween condense into dews. When water drops produced due tothe dew condensation flow and reach to a bottom portion (around thearray attachment portion) of the thermostatic oven 102, it is possiblethat an arc discharge is caused around the array attachment portionwhich is near an electrode being applied of a high voltage. For thisreason, a dew acceptor 303 having a structure like a rain gutter isprovided inside the thermostatic oven 102. The water drops reached tothe dew acceptor 303 are guided therealong to a drain hole 304 providedin the thermostatic oven 102 and is discharged outside the thermostaticoven 102 through a drain (not shown). Thereby, a possible damage of thethermostatic oven 102 by the arc discharge is prevented.

Further, the thermostatic oven 102 is provided with an interlock switch305, and a pin 306 for the interlock switch 305 is attached on the doorat the corresponding position thereto. When the door is closed theinterlock switch 305 is put into a condition of being pushed, thus, thethermostatic oven 102 functions. When the door is opened, a worker cantouch the irradiation and detection portion, thus, the laser beam isautomatically turned off so as to ensure safety. Further, since theworker can touch to the vicinity of the heat source, the power sourcefor Peltier elements is also automatically interrupted so as to ensuresafety which is also effective to protect the Peltier elements. When thedoor is opened during high temperature control of the Peltier elements,the temperature in the thermostatic oven 102 suddenly drops,accordingly, the control performs a heating operation so that thetemperature again restores to the set temperature. Therefore, if thedoor is kept opened, the control unit continues to issue the heatingcommand which possibly damage the Peltier elements due to overloading.With the present embodiment, such accident can be prevented.

The Peltier unit 312 working as a heat source and including the heatradiation fins and the heat radiation fan is contacted to an aluminum(Al) plate 308 at the back face of the thermostatic oven 102. On the Alplate 308 an insulation film is closely adhered so as to prevent an arcdischarge. The Al plate 308 transmits heat from the heat source insidethe thermostatic oven 102 through heat conduction to thereby keep thetemperature inside the thermostatic oven 102 constant. The temperaturein the space determined by the heat from the Al plate 308 which isheated or cooled is stabilized through agitation and circulation of theair inside the thermostatic oven 102 by the fans 309 and 310 disposedtherein. An Al plate 311 is closely adhered to a collecting portion ofthe capillary array directed to the detection portion from thethermostatic oven 102 and is disposed for diffusing heat generated atthe time of high voltage application. The temperature in the space ofthe thermostatic oven 102 is monitored by an in-chamber temperaturesensor 313 and a temperature control is performed based on themonitoring.

FIG. 5 shows a detail of a capillary array holder 1201. Further, FIG. 4shows a detailed structure for attachment between the thermostatic oven102 and the holder 1201. At the bottom portion of the thermostatic oven102 as shown in FIG. 3, a holder 1306 for securing the capillary arrayholder 1201 is provided. The capillary array holder 1201 is providedwith a latches 1303 and 1304 which are to be inserted into attachmentholes 1301 and 1302. An electrode 1305 is provided in a recess 1306 andis connected to an electrode connection portion 1401 as shown in FIG. 5.One ends of the capillaries of the capillary array are respectivelyinserted one by one into holes 1307 of the capillary array holder 1201and are connected to electrodes 1308.

A structure of the capillary array itself is shown in FIGS. 8 and 12.The capillary array will be explained with reference to FIG. 8. Thecapillary array is constituted by an array holder portion 401 whichattaches the capillary array to the thermostatic oven, capillaries 402,a light measurement portion 403, a gel injection portion 404 and anelectrode portion 405. In the present embodiment, an example ofsimultaneous measurement of 16 samples set at a marketed micro-tighterplate having 96 holes or 384 holes is shown. 16 pieces of capillaries402 and electrode portions 405 are included in the capillary array. Amaterial of the capillaries is usually fused quartz and on the surfaceof the capillaries except for the portion where the laser beam isirradiated and fluorescence is detected a high polymer protectivecoating such as polyimide is formed. In order to set the plurality ofcapillaries within the thermostatic oven as illustrated, in that whilepreventing from tangling thereof or from concentrating thereof in abundle shape, a separator 501 as shown in FIG. 9 is used. The separator501 is in a film or plate shape, and at both ends of the separator 501slits 503 for holding the capillaries one by one are formed. Whenhandling a capillary array it is preferable to preserve, manage andhandle the capillary array while attaching the separator 501 as it is.The number of separators 501 can be increased depending on the length ofthe capillary array. For example, for a capillary array of about 36 cmone separator 501 is sufficient, but for a capillary array of about 80cm it is preferable to attach about five separators 501.

The separator 501 is set between the array holder 401 and the lightmeasurement portion 403 and holds the capillaries 502 so as not totangle and concentrate. By means of the holder 401 and the separator501, even if heat is generated from the capillaries due to the highvoltage application on the capillaries during electrophoresis, thetangling and concentration of the capillaries are prevented, the heat isdissipated and a temperature increase in the separation medium duringthe measurement is prevented.

Even if ventilation is improved through the provision of the separators501, since the capillaries are usually not a rigid body but showelasticity, it is important to always dispose the capillaries at apredetermined position in the thermostatic oven even when such ascapillaries are exchanged and an operator is altered. Therefore, aseparator holder 601 as shown in FIGS. 10A and 10B is used at the sideof the thermostatic oven wherein FIG. 10A is a top view thereof and FIG.10B is a side view thereof. The separator 501 is attached from the topas shown in FIG. 10A. As will be seen from FIG. 10B side view, theseparator holder 601 is provided two legs 602 extending therefrom.

An attachment and detachment of the separator holder 601 to thethermostatic oven is performed in the following manner. The leg 602 isinserted into an attachment hole for the separator holder 601 which isformed on the Al plate 308 in the thermostatic oven at a position wherethe separator is to be set, and turns the separator holder 601 by 90°.When projecting the leg portion of the separator holder 601, the shapeof the leg shows an ellipse or a long axis shape, therefore, when theleg is turned by 90°, the long axis direction of the leg becomes inparallel with the short axis of the attachment hole. Since the long axisof the leg portion is longer than the short axis of the attachment hole,the separator holder 601 is secured to the Al plate 308. When detachingthe separator holder 601, the separator holder 601 is turned again by90° so that the long axis of the leg portion becomes in parallel withthe long axis of the attachment hole, thereby, the separator holder 601can be detached from the attachment hole. Thus, the separator holder 601can be attached and detached, easily as explained above.

When using capillary arrays having different length, number ofseparators are prepared depending on the respective length of thecapillary arrays and are set at positions depending on the lengththereof. If the separator holders can not be easily attached or detachedor not totally be attached or detached, a separator holder which is notused for a certain length capillary array may interfere the capillaryarray now used.

FIG. 11 is an imaginary diagram showing layouts when capillary arrays of36 cm, 50 cm and 80 cm are set in the thermostatic oven and positions ofseparator holders used for all of capillary arrays having differentlength. Actually, during one time separation and analysis only onecapillary array is used. Namely, when using the shortest capillary arrayA, the capillary array is shaped in a desirable form, disposed and heldon the inner wall of the thermostatic oven by means of a common arrayholder E, a holder “a” for the array A and a common holder D. Whenholding a comparatively long capillary array B, the capillary array isheld by means of the common holder E, holders b1 and b2 and the commonholder D. When holding the longest capillary array C, the capillaryarray is held by means of the common holder E, holders c1, c2, c3, c4and c5 and the common holder D.

The respective separator holds are set at positions not interruptingwind blow by the fans for circulating air in view of wind blow directionin the thermostatic oven. Further, in order to minimize the number ofholes formed in the Al plate 308, the holes which can be used in commonare increased. In the present embodiment, the position nearest to theseparate holder 601 and the position nearest to the light measurementportion are determined as the common positions. Through the common useof the separators a compact electrophoresis apparatus can be realized.

Since the separator holders are attached and detached depending on therespective length of the capillary array to be used, marks associatingwith the length of array which uses the concerned attachment holes areapplied near the respective separator holder attachment holes on the Alplate 308 or on the insulation film closely adhered to the Al plate 308.Thereby an erroneous operation of attaching a separator holder to anerroneous attachment hole can be avoided.

Now, how the capillary array holder is attached to the thermostatic ovenwill be explained. FIGS. 3 through 5 show a structure and attachmentposition of the capillary array holder 1201. In the drawings, theillustration of the capillary portion in the capillary array is omitted.The thermostatic oven is cut out at a position where the array holder isset so that the face of the thermostatic oven which contacts the doorbecomes flush with the door side of the array holder when the arrayholder is set. Further, when the latches 1303 and 1304 attached to thecapillary array holder 1201 are fitted into the respective attachmentholes 1301 and 1302, the capillary array holder 1201 is secured to thethermostatic oven. This reproducibility with regard to attachmentposition is also important, because the same determines the relativepositional relationship between the capillary array and the autosampler. Therefore, the non-circular recess 1306 is provided on aportion of the electrode 1305 at the array holder attachment portion forthe thermostatic oven. Examples of the non-circular shape are such asellipse and long circle. FIG. 5 is a view when seen the array holder inFIG. 4 from the back face thereof. A projection 1401 is provided at aposition corresponding to the recess 1306 in the thermostatic oven. Whenthe size clearance of these recess and projection at the time of fittingis determined as in the same level as the positional accuracy requiredduring attachment and detachment of capillary array, a reproducibilitywith regard to attachment and detachment position in the directions ofright and left, up and down and rotation can be maintained. A positionalreproducibility in backward direction is maintained through contactbetween the reference face 1402 of the capillary array and the opposingface of the thermostatic oven.

Further, a high voltage of more than 15 kV is applied to the electrode1305, around the electrode there is provided a recess and an insulativerubber is laid out over the recess so as to closely contact with theprojection 1401 in the capillary array holder 1201. Thereby, an air gapbetween the electrode 1305 and the capillary array holder 1201 iseliminated. Further, through the fitting structure between the recessand the projection a creeping distance from the high potential portionto the grounding portion is prolonged, thereby, a possible arc dischargeis suppressed.

FIG. 6A is a schematic structure of the thermostatic oven shown in aplane cross sectional view and FIG. 6B in a side cross sectional viewthereof. The Al plate 1501, which uniformly transfer heat from thePeltier elements working as a heat source into the thermostatic oven andstabilizes the temperature therein, is contacted to a Peltier unit 1508from the back face of the thermostatic oven through a Peltier contactportion 1502. A reason of using a Peltier unit is that the Peltier unitcan set not only a higher temperature than the room temperature but alsocan set a lower temperature than the room temperature. The heattransferred from the Peltier unit to the Al plate 1501 is transferred tothe air in the thermostatic oven and the air is agitated and circulatedby the fans 1503 and 1504 which further stabilizes the temperature ofthe thermostatic oven. The temperature in the thermostatic oven ismonitored by a temperature sensor 1505.

The circumference of the thermostatic oven is covered by a heatinsulating material 1506 and the heat flow to and from the outside ofthe thermostatic oven is interrupted. Further, with a Peltiertemperature sensor 1507 the temperature of the Peltier unit ismonitored.

FIGS. 7A and 7B show air suction and below out directions of two flatfans used in the thermostatic oven. As illustrated in the drawingsthrough the use of a flat fan of which air suction and blow outdirections are not in parallel, the thickness of the thermostatic ovenis reduced. FIGS. 7C and 7D are views when seen these fans from the backface thereof. An example when the fans are disposed as in FIGS. 7A and7B is shown in FIGS. 6A and 6B. FIG. 6A shows the position where thefans are disposed, and is a cross sectional view seen from the arroweddirection in FIG. 6B.

When disposing the fans as illustrated in FIG. 6B, the air suction portis located at the side of the door of the thermostatic oven, in that atthe position remote from the heat source. If the fans are located at thewall face in contrast to the arrangement in FIG. 6B, the air suctionport locates at the opposite side of the door, in that at a positionnear the heat source. When the air is sucked near from the heat source,the air having temperature near the heat source other than the currenttemperature in the thermostatic oven can be circulated which isdesirable. In an actual arrangement, the fans can be secured at the doorside, however, if the fans can not be secured at the door side becauseof problems such as wiring, the fans can be disposed at the oppositeside of the door through provision of such as a spacer and a holder atthe side thereof. In the example as illustrated in FIG. 3, the fans aredisposed at the opposite side of the door. As illustrated in FIG. 7A,the air blow out direction of the fan is offset from the center axis ofthe air suction port. Further, if it is difficult to align all of theair blow out directions in a target direction depending on the structureof fans, it is not necessary to locate all of the air suction ports ofthe plurality of fans at the side of the heat source, but a part of theair suction ports can be located at the side of the heat source. In theexample of FIG. 3, the air suction port of the fan 309 is located at theside of the heat source, but the air suction port of the fan 310 is atthe side of the door.

Further, in view of the air circulation, a fan with a large air capacityis effective because of no air holdup, however, if the capillary arrayis caused to vibrate because of the large air capacity which will leaddeterioration in separation through the electrophoresis. Therefore, afan having a large air capacity is disposed at a portion where novibration affects to the capillary array and a fan having an aircapacity causing no vibration is selected for a portion where vibrationaffects to the capillary array. In FIGS. 6A and 6B, the air capacity ofthe fan 1504 is selected smaller than that of the fan 1503.

In an actual temperature control, a method in which only the airtemperature in the thermostatic oven is monitored and a feed backsignals are applied to the output of the Peltier unit is generally used.However, in addition to the temperature sensor 1505 for the thermostaticoven, the temperature sensor 1507 for the Peltier unit can be used forthe temperature control. FIG. 17 shows a block diagram for explaining anexemplary temperature control which is an example of a proportional andintegration control. In the drawing K1 through K5 are control constants,and +Toff is a correction constant which is used and processed by asoftware when read indication values from the respective sensors includeoffset and usually zero. K1 is a proportional coefficient. K2·Σ is anintegration term which represents that after multiplying integrationconstant K2 an integration is performed.

The a final resultant output of the control is the Peltier out, andresponse of the Peltier temperature to the Peltier output is quick anddirect. On the other hand, the response of the temperature in thethermostatic oven to the Peltier output is slow and indirect. Therefore,when the Peltier temperature is used for the control, a long waviness oftemperature time constant can be reduced in comparison with when onlythe temperature of slow response in the thermostatic oven is used forthe control which is desirable for the present control.

In the present invention, all of the three functions necessary for acapillary array used for a capillary array electrophoresis apparatus, inthat a buffer liquid injection port to be installed at a buffer liquidcontainer, a light detection portion where laser beam is irradiated andfluorescence is detected and a specimen introduction portion into whicha sample is introduced and to which a voltage necessary for causingelectrophoresis is applied, are used.

As illustrated in FIG. 8, the capillary array electrophoresis apparatusis constituted by a capillary array 402, an injection port 404 of bufferliquid and electrophoresis medium, a light detection portion 403 and asample introduction portion 405. Further, an electrode is secured tocapillaries at the sample introduction portion 405 of the capillaryarray.

By making use of a second syringe 410 in a polymer solution pump, i.e. agel injection pump system 414 which is shown in a simplified form,electrophoresis medium and buffer liquid are injected under pressurefrom a buffer liquid reservoir 412 to the gel injection portion 404 inthe capillary array 402. A high voltage is applied between an electrode407 provided in the buffer liquid reservoir 412 and the electrodeportion 405 of the capillary array.

An entire layout and operation of an electrophoresis apparatus using acapillary array according to the present invention will be explainedwith reference to FIG. 12. The capillary array according to the presentinvention includes a buffer liquid injection port 3010 which is formedby bundling a plurality of capillaries at one ends thereof and is set toa buffer liquid container 3117 for injecting buffer liquid 3136, and aportion where the coating of the plurality of capillaries is removed ofwhich details are illustrated in FIG. 13.

The coating removed portion of the capillaries is arranged in a planeand held on a holder substrate 4005. The holder substrate 4005 isprovided with a window 4011 for passing detection light at a portioncorresponding to the coating removed portion of the respectivecapillaries. The holder substrate 4005 further includes a lightdetection portion provided with a light shielding region defining thewindow for passing the detection light.

In FIG. 12, at the other ends of the capillaries, a sample introductionportion 3032 is constituted into which a sample marked by a fluorescentmaterial so as to supply the fluorescent sample into the capillaryarray, and near the respective capillaries at the top end of thefluorescent sample introduction portion an electrode (not shown) isprovided onto which a voltage necessary for electrophoresis is applied.The voltage necessary for the electrophoresis is applied between theelectrode provided at the capillary array holder 3030 and a reservoir3117 for supplying the electrophoresis medium from a power source 3119.

As illustrated in FIG. 12, the capillary array electrophoresis apparatusis constituted by such as a sample measurement portion 3116, a bufferliquid container 3117, a fluorescent sample container 3118, a highvoltage power source 3119, an optical system comprising a laser beamsource 3120, a mirror 3121, a beam splitter 3122, a condenser lens 3123,a first lens 3124, an optical filter and transmission type grating 3125,a second lens 3126, and a CCD camera 3127, and a processing unit 3128.The sample measurement portion 3116 is constituted by capillaries 3001,a light detector, i.e. a light detection portion 3020, a buffer liquidinjection port 3010 and a conductive fluorescent sample injection port3032.

Now, the operation principle of the capillary array electrophoresisapparatus will be explained with reference to FIG. 12. The laser beam3133 generated from the laser beam source 3120 is divided into two partsby the beam splitter 3122 and the advancing direction thereof is changedby the mirror 3121. The laser beam 3133 is condensed by the condenserlens 3123 and is irradiated to the capillaries 3001 from a direction inparallel with the alignment direction of the capillaries 3001. Theinside of the capillaries 3001 is filled with the sample marked by afluorescent material (fluorescent sample 3134), and when the laser beam3133 is irradiated, the fluorescent sample 3134 emits fluorescence 3135.For the detection of the fluorescence 3135, the fluorescence 3135emitted in substantially perpendicular direction with respect to thealignment plane of the capillaries 3001 is converted into parallel lightby the first lens 3124, is effected of image/color division by theoptical filter and transmission type grating 3125, and thereafterimage-formed on the CCD camera 3127 by the second lens 3126 and isdetected by the CCD camera 3127. The detected measurement data isprocessed by the processing unit 3128.

In FIG. 12, the laser beam 3133 is irradiated from the both sides of thelight detection portion 3020, however, the apparatus can be constitutedin such a manner that the laser beam 3133 is irradiated only from oneside thereof. Further, the layout of the light receiving optical systemis not limited to that illustrated in FIG. 12. Still further, the numberof constituting capillaries is not limited to 16 pieces and thestructure of the buffer liquid injection port 3010 and the conductivefluorescent sample injection port 3032 is not also limited to thoseshown in FIG. 12.

Now, an operation sequence of the capillary array electrophoresisapparatus will be explained. The buffer liquid 3136 contained in thebuffer liquid container 3117 is injected into the capillaries 3001 fromthe buffer liquid injection port 3010. Subsequently, the conductivefluorescent sample injection port 3032 is immersed in the fluorescentsample container 3118 filled with the fluorescent sample 3134 and thefluorescent sample 3134 is injected into the capillaries 3001.Thereafter, the conductive fluorescent sample injection port 3032 isimmersed in a buffer liquid container (not shown) containing bufferliquid, and a high voltage is applied between the buffer liquidinjection port 3010 and the fluorescent sample injection port 3032 bythe high voltage power source 3119 to thereby cause electrophoresis inthe capillaries. Since the moving speed by electrophoresis isproportional to the electric charge magnitude of the molecules and isreverse proportional to the mass of the molecules, the fluorescentsample 3134 is separated. Through continuous application of the highvoltage for a long time the electrophoresis is caused for a long timeand the fluorescence 3135 emitted at this time is continuously measured.

The sample introduction portion 3030 is structured by insertingcapillaries into stainless tubes. Respective stainless tubes 3321 aresoldered to an electrode plate with a protective cover and throughapplication of a voltage to a connecting portion 3031, the voltage isapplied to all of the stainless tubes. As has been explained, since thecapillary array itself is provided with all necessary functionsincluding the buffer liquid injection port 3010 attached to the bufferliquid container 3117, a light detection portion 3020 in which laserbeam is irradiated and the fluorescence is detected and the sampleintroduction portion 3030 through which the fluorescent sample 3134 isintroduced and a voltage necessary for electrophoresis is appliedthereto, when an exchange of the capillary array is required, thecapillary array can be exchanged with a very easy handling.

Further, the top of the fluorescent sample injection port 3032 is sealedby an adhesive so as to prevent carry over of such as the sample. A kindof the adhesive used is an epoxy series adhesive and the same is fullycured so as not to affect the electrophoresis. Gaps between capillaries3001 and insertion portions 3033 therefor in the sample introductionportion 3030 and between the fluorescent sample injection port 3032 andthe protective covers are sealed with an adhesive. Thereby, a possibleelectric insulation reduction is prevented which can be caused whenwater contained in the sample and the buffer liquid penetrates into thecovers of the stainless tubes.

When once detaching the capillary array from the apparatus and storingthe same after measurement of the sample, a dry preventive containercover (not shown) is attached so as not to dry the buffer liquid 3136.The container cover is a dry preventive cover for the sampleintroduction portion 3030. The container cover is attached to the sampleintroduction portion 3030 while charging pure water therein. Thecontainer cover is provided with an O ring to thereby prevent a possibledrying. It is also effective to provide a dry protective cap (not shown)for the buffer liquid injection port 3010. In such instance the cap isset onto the buffer liquid injection port 3010 under the condition inwhich a small amount of pure water is likely filled therein. When theinner diameter of the cap is determined smaller than the outer diameterof the buffer liquid injection port 3010 by about 5˜15% to therebyprevent a possible drying. As a material of the cap silicon rubber ispreferable, because the silicon rubber causes no adverse effect to thebuffer liquid and the electrophoresis. These cover and cap also work toprotect the top end thereof and to prevent contamination thereof whenshipping the capillary array to a customer.

Each of the capillaries 3001 used in the capillary array as explainedabove is a fused quartz tube having inner diameter of 50±10 μm and outerdiameter of 340±20 μm. Since the fused quartz tube itself breaks veryeasily, a polyimide coating having thickness of 15±5 μm is applied onthe surface of the capillary. In view of limiting the amount offluorescent sample 3134 it is desirable to reduce the inner diameter ofthe capillary, however, on the other hand in view of a concave lenseffect due to refractive index difference between the fluorescent sample3134 and fused quartz, the capillary having a too small inner diametermakes the measurement difficult. Therefore, the inner diameter of 50˜100μm is preferable for the fused quartz tube. Further, in order tosuppress the above refractive index difference it is preferable that theouter diameter of the fused quartz tube is small, however, a too smallouter diameter makes assembling thereof difficult because of staticelectricity, therefore, the outer diameter of 250˜350 μm is preferablefor the fused quartz tube. The coating material for the capillary 3001is not limited to the polyimide, a material having an equivalentelectrical insulation and other properties as those of polyimide can beused.

FIG. 13 is an exploded view of a structure of an irradiation anddetection portion in a capillary array used for the present invention.Glass substrate 4023 includes a groove 4011 for laser irradiation and ablack coating 4046 on the back face of the substrate. The surface of theglass substrate 4023 where the polyimide coating contacts is processedin such a high accuracy that interference fringes can be observed on thesurface, and the flatness degree thereof is high.

A plurality of a capillaries 4001 are contacted to the highly flattenedsurface via the polyimide coating and are aligned thereon. Thereby, theplurality of the capillaries 4001 follow the glass substrate 4023 andare aligned thereon with high accuracy and easily.

The polyimide coating of the capillaries at the irradiation anddetection portion is removed to constitute a transparent portion 4009.The removal can be performed, for example, in such a manner that afterremoving the polyimide coating by a predetermined size one by oneseparately, then the removed portions are arranged. However, when thepolyimide coating is removed one by one by a predetermined removingwidth, a processing error is caused and the removed width varies.Further, the arrangement is performed in such a manner that the removedportions, in particular, the boundaries (the boundary where thepolyimide resin 4010 is cut out) align each other, however, suchoperation likely causes error and takes time.

Usually, a non alignment of the boundary portion can be immediatelyrecognized. In the worst case, a remaining polyimide resin can beobserved from a through window 4006 which causes great adverse effect tothe detection.

Therefore, instead of the one by one coating removal, after arrangingthe plurality of capillaries when the polyimide coating is removedcollectively, the removed portions of polyimide resin 4010 on theplurality of capillaries are neatly aligned. It is easily recognizedwhich aligning method is used when observing the alignment of theboundaries. The predetermined width and the predetermined position ofthe polyimide resin removed position can be freely changed inclusivelywith the plurality of the capillaries.

The capillaries 4001 are sandwiched by the glass substrate 4023 for theirradiation and detection portion and an opposing member 4002. Theopposing member 4002 is provided with the detection through window 4006and fluorescence is emitted from a transparent capillaries 4007. Grooves4008 which press capillaries inner face of the opposing member 4002.

When no black coating 4046 is provided, the laser beam penetrates andpasses through the plurality of capillaries which are aligned in a highaccuracy. At this moment, scattered light from the surface of thecapillaries passes through the glass substrate 4023 and is irradiated toa fluorescent emitting material on the surface of the opposing member4002 disposed opposite to the glass substrate 4023, and the fluorescenceemitted thereby returns to the capillaries, further passes the throughwindow 4006 and is directed to the first lens which causes noises.Further, when a fluorescent emitting material is deposited on the backface of the glass substrate 4023, such likely causes noises.

However, when the black coating 4046 is applied on the back face of theglass substrate 4023, even if a fluorescent emitting material iscontained in the opposing member 4002 and further a fluorescent emittingmaterial is deposited after the black coating 4046 is applied, thescattered light is absorbed by the black coating 4046, thereby, thecauses of noises are removed. As a material of the black coating a paintwhich emits no fluorescence is used. As a typical paint application worka sick screening is used, however, other painting method can be used,and further a manual painting can also be used.

An optical system according to the present invention will be explained.In an embodiment according to the present invention, laser beam isirradiated to one or both end sides capillaries in the capillary arrayconstituted by a plurality of capillaries arranged on one flat plane,the laser beam successively propagates the adjacent capillaries andcrosses the capillary array, and a CCD camera is used as a fluorescentdetection means in the capillary array electrophoresis apparatus. Inthis instance, the alignment portion of the capillary array constitutingthe irradiation and detection portion is disposed in parallel with thelaser beam irradiation direction. More specifically, the irradiation anddetection portion is placed vertically, and the laser beam is irradiatedto the irradiation and detection portion from upper direction or fromupper and lower directions after dividing the laser beam into twoportions. FIG. 8 shows such arrangement. Further, for the sake ofillustration, the irradiation and detection portion is disposedhorizontal in FIG. 12, however, the same is actually disposed verticallyas in FIG. 8 and the laser beam is irradiated onto the irradiation anddetection portion of the capillary array from upper and lower directionsafter being divided into two portions. Such arrangement is suitable forreducing the size of the optical system for the irradiation anddetection. With this arrangement in connection with the laser beam, thepassage of the laser beam is constituted so as not to direct to the sidewhere an operator usually works which enhances safety.

A light emission intensity from a single capillary is determined as onewhich is detected by a number of CCD pixels nearest to the full width athalf maximum in an emitted light distribution curve of the capillary inthe capillary alignment direction with respect to the formed image onthe CCD camera. Further, the optical system further includes a rotationangle adjustment function having a rotation angle accuracy of about 0.1°around the optical axis for the CCD camera and the grating.

In a capillary electrophoresis DNA sequencer, when causingelectrophoresis of a sample through a high voltage application onto thecapillaries, whether the electrophoresis is correctly performed isjudged by monitoring a current flowing through the capillary. Generally,such monitoring is performed by a built-in meter in the high voltagepower source, however, when an insulation around the high voltageelectrode is poor, a leakage current is caused, thereby, the meterindicates a current value including the leakage current other than thecurrent actually flowing through the capillary which is one ofdrawbacks. Therefore, as illustrated in FIG. 16, a resistor R isinserted between the end of the capillary and the ground, and only thecurrent flowing the capillary is correctly monitored through measurementof a voltage between the resistor R, thereby, whether theelectrophoresis is correctly performed is correctly judged.

When effecting a capillary electrophoresis, it is necessary to apply avoltage from several kV to several 10 kV to a sample. In the presentinvention, in order to fulfill the above necessity a method in which aneedle is inserted together with a capillary into the sample from thecapillary holder is used. When an insulation between the needle and asurrounding metal portion is poor, a discharge from the high voltageportion is induced which prevents a correct measurement. In order toenhance the insulation property, it is conceived to increase thedistance between the needle and the surrounding metal portion dependingon the applied voltage, however, such measure is structurally limited.In the present invention, with regard to the portions which come closeinherently because of structure reason, an enclosed structure with aninsulation material is used. Further, a portion which applies a highvoltage to the capillary holder is made detachable, and thus in order torealized the enclosed structure a plug-in structure is employed and theclosedness is achieved by a rubber.

A unit pump in an embodiment according to the present invention will beexplained with reference to the drawing. FIG. 14 is a schematic diagramof an electrophoresis apparatus with a gel injection mechanismrepresenting an embodiment according to the present invention.

A capillary array 8118 is constituted by at least two capillaries andone ends thereof are inserted into a block 8116 and the other endsthereof are integrated with an electrode which is connected to a powersource 8121.

Prior to a measurement, gel serving as a separation medium or anelectrophoresis medium is charged into the capillary array 8118 from theside of the block 8116. To the block 8116 an injection use syringe 8113which injects gel into the capillary array and a refill use syringe 8114for refilling gel into the injection use syringe 8113 are attached. Thevolume of the refill use syringe 8114 serving as a first syringe islarger than the volume of the injection use syringe 8113 serving as asecond syringe. Further, the volume of the second syringe is basicallydetermined as one which can supply an amount of gel polymer for one timeseparation and analysis. In the block 8116, a first flow passagecommunicating between the refill use syringe 8114 and the injection usesyringe 8113 and a second flow passage communicating between theinjection use syringe 8113 and the capillary array 8118 are formed.Further, the second flow passage is provided with a branch passage to abuffer reservoir 8126 which is kept at the ground potential duringelectrophoresis.

Further, in the flow passage communicating between the refill usesyringe 8114 and the injection use syringe 8113 a check valve 8115 isinserted so as to prevent a reverse flow of gel to the refill usesyringe 8114. The refill use syringe 8114 and the injection use syringe8113 are pressed by respective drive units 817 and 818 which arecontrolled by a control unit 812. To the drive units 817 and 818respective linear encoders 819 and 8110 are installed, and throughreading values indicated by the linear encoders 819 and 8110 positionalinformation of the drive units 817 and 818 is transferred to a computer811 via the control unit 812.

After being filled with gel, the capillary array 8118 is moved to asample container 8122, and after sucking in the sample through anelectrical action, the capillary array 8118 is moved to a buffer vessel8123. When a voltage is applied via the electrode portion for thecapillary in the buffer vessel 8123, an electric field is induced in thecapillaries and the introduced sample begins electrophoresis. Because ofdifference in electrophoresis speed depending on such as molecularweight of the introduced sample, a separated sample can be detected at adetection portion 8117. When completing an analysis, the inside thecapillary array 8118 is replaced by a new gel by the injection usesyringe 8113 and again the following measurement is started.

FIG. 15 shows a cross sectional view of a connecting portion between thecapillary array and the pump system. In order to prevent an invasion ofbubbles into the capillaries from the connecting portion, the presentstructure is provided with a bubble vent structure 8201 at a flowpassage 8202 in the pump system. Further, through shaping the top end ofa ferrule 8205 into WD type (a long circle type), a simultaneousrotation of the ferrule 8205 when fastening a push screw 8204 isprevented. Further, through elongation of the ferrule 8205 so as toextend from the push screw, an exudation of the gel is prevented. Stillfurther, through forming the sleeve independent, exchange thereof isenabled.

When a refillable gel is used, it is necessary that the pressures atboth ends of the capillary are kept equal so as not to move the geltherein. Therefore, the liquid surfaces of the buffer vessels for bothcathode and anode have to be kept at the same levels. In the presentembodiment, since the block is divided into upper and lower blocks,therefore, the lower block serving as a buffer vessel is arranged toassume the same height as the other buffer vessel and the other upperblock is disposed at a position where the gel is injected easily(actually at a position where the shortest capillary can reach).

FIG. 18 shows a flow chart for a gel injection work. The control unit812 which has received a gel injection command from a computer 811 atfirst performs an initial operation for the gel injection unit (step202) and closes the buffer valve 8124 (step 203). Thereafter, the driveunit 817 moves down and the position of the plunger 8111 for theinjection use syringe 8113 is automatically detected (step 204).Subsequently, at the side of the refill use syringe 8114 the drive unit818 moves down to the position of the plunger 8112 (step 205).Thereafter, based on the value of the injection side linear encoder 819the amount of remaining gel in the injection use syringe 8113 isconfirmed for the first time (step 206). When the gel in the injectionuse syringe 8113 is short, prior to gel injection, gel refillingoperation from the refill use syringe 8114 to the injection use syringe8113 is performed.

The gel refilling operation is performed as follows. At first, based onthe value of the refill side linear encoder 8110 a gel remaining amountis confirmed (step 212). When the remaining amount is sufficient, theinjection side drive unit 817 moves to a plunger position where theinjection use syringe 8113 is full (step 213), thereafter apressurization for the refill use syringe 8114 by the drive unit 818 isstarted (step 214). At this moment, almost all gel pushed out from therefill use syringe 8114 and flown into the block 8116 flows into theinjection use syringe 8113 while lifting the plunger 8111 for theinjection use syringe 8113, not into the capillaries because ofdifference in flow passage resistance. When the injection use syringe8113 is filled, the plunger 8111 hits the injection side drive unit 817to prevent further gel refilling. At this instance, the control unit 812confirms periodically (for example, an interval of 1 sec.) the value ofthe encoder for the drive unit 818, and if no change is observed for 5sec. in that if the drive unit 818 does not move, the control unit 812judges that the gel refilling operation has been completed and stops themotor (step 215). After completing the gel refilling, the refill sidedrive unit 818 moves upward so as to release the pressure and waits forthe subsequent command (step 216). If the remaining amount of the refilluse syringe 8114 is short, a message of remaining amount shortage isdisplayed on the screen of the computer 811, in such instance afterrefilling the gel into the refill use syringe 8114 through manualoperation by a user, the operation is restarted (step 217).

When the remaining gel amount in the injection use syringe 8113 issufficient or after the gel refilling has been completed, the injectionside drive unit 817 starts to press the injection use syringe 8113 andthe gel injection into the capillaries 8118 is started (step 207). Atthis moment, the check valve 8115 prevents a gel reverse flow into therefill use syringe 8114 and further, since the buffer valve 8124 isclosed, the pushed out gel from the injection use syringe 8113 flowsinto the capillaries which are only flowable passages. When apredetermined amount of gel is charged, the injection side drive unit817 stops the pressurization (step 208) and moves upward so as torelease the pressure and waits for the following command (step 209).Subsequently, after the buffer valve 8124 is opened (step 210), avoltage is applied to the electrode for the capillaries to start anelectrophoresis (step 211).

In a DNA sequencer, the gel in the capillaries has to be refilled forevery measurement, therefore, it is necessary to generate a highpressure for injecting the gel having a higher viscosity into thecapillaries and to ensure a capacity of carrying out a continuousinjection. However, both the cross section of a piston and the capacityof a high pressure resistant syringe are generally small. Namely, sinceit was difficult to obtain a syringe which satisfies the bothrequirements with regard to high pressure resistance and large capacityat the same time, in the present invention a combination of theinjection use syringe having a high pressure resistance and the refilluse syringe having a large capacity is employed. Further, in order toperform the injection and refilling automatically the structure makinguse of the check valve is necessitated. Further, when the gel in therefill use syringe becomes empty, the gel refilling is manuallyperformed by a user, in this instance the syringe is detached forrefilling. After attaching the syringe, the apparatus according to thepresent invention automatically recognizes the position of the plungerfor the syringe through the plunger position detecting function,therefore, an automatic continuous measurement can be effectedimmediately after the user attaches the syringe to the apparatus.

As a measure of safety operation according to the present invention, ashutter for laser beam is constituted as shown in FIGS. 19A and 19B inwhich a metal plate shutter 903 is attached to a reversible steppingmotor, two shock absorbing rubbers 901 and 902 are disposed within arange of the shutter reciprocating movement and the shutter 903 hits tothe shock absorbing rubbers at the time of open and close and stopsthere. Further, the attachment angle in rotational direction of theshutter to the motor is adjusted so that the shutter does not cross thevertical axis during the open and close operation thereof.

With the shutter which is constituted by attaching the metal plate tothe reversible stepping motor, a vibration is caused during opening andclosing of the shutter. As a result, even under a closed condition thelaser beam leaks the shutter and causes a fluctuation in exposure timeor abnormality when reading CCD signals. Further, depending theattachment angle to the motor the shutter can not keep closed conditionduring turning off of the power source due to the weight thereof.

When a part of a plane of the glass base at the side where the capillaryarray is aligned is pressed to all of or a part of an array attachmentreference plane in the optical system, the array attachment referenceplane representing the reference plane at the side of theelectrophoresis apparatus coincides with the glass base representing thereference plane at the side of the capillary array.

At the side of the electrophoresis apparatus the position of the laserbeam with respect to the array attachment reference plane is determinedat a positional accuracy below 10 μm. When determining the position ofthe laser beam with the laser beam condenser lens, the position of thelaser beam condenser lens with respect to the array attachment referenceplane is determined at a positional accuracy below 10 μm. In thisinstance, the laser beam which makes incident to the lens with apredetermined angle can be irradiated at a predetermined position on thecapillary array.

At the side of the capillary array through pressing the capillaries onto the glass substrate, the position of the capillaries with respect tothe glass substrate is determined at a positional accuracy below 10 μm.Accordingly, by pressing the array attachment reference plane to theglass base, the positions of the capillaries and the laser beams can bedetermined at a positional accuracy of about 10 μm with a goodreproducibility.

According to the present invention, an electrophoresis apparatus inwhich capillary arrays having a variety of length can be easilyexchanged and held and further can be easily adapted to a variety ofmodifications of separation and analysis objects.

1. A capillary array electrophoresis apparatus comprising: a pluralityof capillaries, each of which includes a first terminal portion and asecond terminal portion; a first buffer vessel containing first bufferliquid into which the first terminal portions of the plurality ofcapillaries are immersed; a flow passage of which one end is connectedto the second terminal portions of the plurality of capillaries; asecond buffer vessel containing second buffer liquid to which the otherend of the flow passage is contacted; a power source which applies avoltage to the first buffer liquid and the second buffer liquid; anexcitation light system which irradiates excitation light to thecapillaries; and a light receiving optical system which detectsfluorescence; wherein, a liquid level of the first buffer liquid andthat of the second buffer liquid are substantially the same, and whereinthe second terminal portions are located at a higher position than theliquid level of the first buffer liquid and that of the second bufferliquid.
 2. A capillary array electrophoresis apparatus according toclaim 1, wherein a capillary arrangement direction at the first terminalportions is substantially perpendicular to a capillary arrangementdirection at the second terminal portions.
 3. A capillary arrayelectrophoresis apparatus according to claim 1, wherein a capillaryarrangement direction at the first terminal portions is substantiallyvertical and a capillary arrangement direction at the second terminalportions is substantially horizontal.
 4. A capillary arrayelectrophoresis apparatus according to claim 1, wherein a crosssectional area of the flow passage is larger than the total sum of crosssectional areas of the plurality of capillaries.
 5. A capillary arrayelectrophoresis apparatus comprising: a plurality of capillaries, eachof which includes a first terminal portion and a second terminalportion; a first buffer vessel containing first buffer liquid into whichthe first terminal portions of the plurality of capillaries areimmersed; a flow passage of which one end is connected to the secondterminal portions of the plurality of capillaries; a second buffervessel containing second buffer liquid to which the other end of theflow passage is contacted; a power source which applies a voltage to thefirst buffer liquid and the second buffer liquid; an excitation lightsystem which irradiates excitation light to the capillaries; and a lightreceiving optical system which detects fluorescence; wherein, a liquidlevel of the first buffer liquid and that of the second buffer liquidare substantially the same, and wherein a filling mechanism which fillsfilling medium into the capillaries through the second terminal portionsis connected to the flow passage.
 6. A capillary array electrophoresisapparatus comprising: a plurality of capillaries, each of which includesa first terminal portion and a second terminal portion; a first buffervessel containing first buffer liquid into which the first terminalportions of the plurality of capillaries are immersed; a flow passage ofwhich one end is connected to the second terminal portions of theplurality of capillaries; a second buffer vessel containing secondbuffer liquid to which the other end of the flow passage is contacted; apower source which applies a voltage to the first buffer liquid and thesecond buffer liquid; an excitation light system which irradiatesexcitation light to the capillaries; and a light receiving opticalsystem which detects fluorescence; wherein, a liquid level of the firstbuffer liquid and that of the second buffer liquid are substantially thesame; and further comprising a thermostatic oven from which the firstterminal portions and the second terminal portions are projected.
 7. Acapillary array electrophoresis apparatus comprising: a plurality ofcapillaries, each of which includes a first terminal portion and asecond terminal portion; a first buffer vessel containing first bufferliquid into which the first terminal portions of the plurality ofcapillaries are immersed; a flow passage of which one end is connectedto the second terminal portions of the plurality of capillaries; asecond buffer vessel containing second buffer liquid to which the otherend of the flow passage is contacted; a power source which applies avoltage to the first buffer liquid and the second buffer liquid; anexcitation light system which irradiates excitation light to thecapillaries; and a light receiving optical system which detectsfluorescence; wherein, a liquid level of the first buffer liquid andthat of the second buffer liquid are substantially the same; and furthercomprising a thermostatic oven having a bottom from which the firstterminal portions are projected and a side from which the secondterminal portions are projected.
 8. A capillary array electrophoresisapparatus comprising: a plurality of capillaries, each of which includesa first terminal portion and a second terminal portion; a first buffervessel containing first buffer liquid into which the first terminalportions of the plurality of capillaries are immersed; a flow passage ofwhich one end is connected to the second terminal portions of theplurality of capillaries; a second buffer vessel containing secondbuffer liquid to which the other end of the flow passage is contacted; apower source which applies a voltage to the first buffer liquid and thesecond buffer liquid; an excitation light system which irradiatesexcitation light to the capillaries; and a light receiving opticalsystem which detects fluorescence; wherein, the second terminal portionsare located at a higher position than the first terminal portions, andwherein the second terminal portions are located at a higher positionthan the liquid level of the first buffer liquid and that of the secondbuffer liquid.
 9. A capillary array electrophoresis apparatus accordingto claim 8, wherein a capillary arrangement direction at the firstterminal portions is substantially perpendicular to a capillaryarrangement direction at the second terminal portions.
 10. A capillaryarray electrophoresis apparatus according to claim 8, wherein acapillary arrangement direction at the first terminal portions issubstantially vertical and a capillary arrangement direction at thesecond terminal portions is substantially horizontal.
 11. A capillaryarray electrophoresis apparatus according to claim 8, wherein a crosssectional area of the flow passage is larger than the total sum of crosssectional areas of the plurality of capillaries.
 12. A capillary arrayelectrophoresis apparatus comprising: a plurality of capillaries, eachof which includes a first terminal portion and a second terminalportion; a first buffer vessel containing first buffer liquid into whichthe first terminal portions of the plurality of capillaries areimmersed; a flow passage of which one end is connected to the secondterminal portions of the plurality of capillaries; a second buffervessel containing second buffer liquid to which the other end of theflow passage is contacted; a power source which applies a voltage to thefirst buffer liquid and the second buffer liquid; an excitation lightsystem which irradiates excitation light to the capillaries; and a lightreceiving optical system which detects fluorescence; wherein, the secondterminal portions are located at a higher position than the firstterminal portions, and wherein a filling mechanism which fills fillingmedium into the capillaries through the second terminal portions isconnected to the flow passage.
 13. A capillary array electrophoresisapparatus comprising: a plurality of capillaries, each of which includesa first terminal portion and a second terminal portion; a first buffervessel containing first buffer liquid into which the first terminalportions of the plurality of capillaries are immersed; a flow passage ofwhich one end is connected to the second terminal portions of theplurality of capillaries; a second buffer vessel containing secondbuffer liquid to which the other end of the flow passage is contacted; apower source which applies a voltage to the first buffer liquid and thesecond buffer liquid; an excitation light system which irradiatesexcitation light to the capillaries; and a light receiving opticalsystem which detects fluorescence; wherein, the second terminal portionsare located at a higher position than the first terminal portions; andfurther comprising a thermostatic oven from which the first terminalportions and the second terminal portions are projected.
 14. A capillaryarray electrophoresis apparatus comprising: a plurality of capillaries,each of which includes a first terminal portion and a second terminalportion; a first buffer vessel containing first buffer liquid into whichthe first terminal portions of the plurality of capillaries areimmersed; a flow passage of which one end is connected to the secondterminal portions of the plurality of capillaries; a second buffervessel containing second buffer liquid to which the other end of theflow passage is contacted; a power source which applies a voltage to thefirst buffer liquid and the second buffer liquid; an excitation lightsystem which irradiates excitation light to the capillaries; and a lightreceiving optical system which detects fluorescence; wherein, the secondterminal portions are located at a higher position than the firstterminal portions; and further comprising a thermostatic oven having abottom from which the first terminal portions are projected, and havinga side from which the second terminal portions are projected.